A fully automated phosphopeptide purification system for large-scale phosphoproteome analysis

作者：Iwase Yuka; Honma Satomi; Matsuzaki Megumi; Miyakawa Yukiko; Kanno Takuma; Ishii Kohei; Furuichi Naotaka; Furukawa Kazuhiro; Horigome Tsuneyoshi^*

来源：Journal of Biochemistry, 2010, 147(5): 689-696.

DOI：10.1093/jb/mvp211

• phosphopeptide
• phosphoproteome
• phosphorylation
• proteomics
• titania

For large-scale phosphoproteome analysis based on mass spectrometry, a fully automated phosphopeptide purification system is essential to obtain reproducible results. An automated system involving pre-cleaning of a sample with a polymer-based reversed-phase column, phosphopeptide purification with a titania column and analysis of the phosphopeptide fraction with a reversed-phase column was developed, and then the analytical conditions for a complex peptide mixture were optimized. A lower flow rate for application of samples to the titania column was essential to obtain high recoveries of phosphopeptides from complex protein digests. Washing with 1 M NaCl and 2-propanol, and two cycles of washing with four solvents for the titania column were necessary to minimize non-phosphorylated peptides in the phosphopeptide fraction. Using this system under the optimized conditions, a peptide fraction including > 90% phosphopeptides could be obtained highly reproducibly from a tryptic digest of a complex protein mixture, i.e. a Xenopus egg cytosol fraction, without any pre-treatment.

• 出版日期2010-5